A method for the synthesis of semaglutide

Abstract

The application discloses a synthesis method of semaglutide, and comprises the following steps: preparing compound 2: taking Arg34GLP-1(9-37) as a starting material, performing a fatty chain modification on Lys at the 26th position of Arg34GLP-1(9-37) by using compound 1, controlling the pH of the reaction, and performing an acylation reaction to obtain compound 2; preparing compound 4: performing an acylation coupling reaction on the terminal amino group of Arg34GLP-1(9-37) in compound 2 by using compound 3 after activation, a condensation reagent and an alkali to obtain compound 4; and preparing semaglutide: taking compound 4 as a raw material, and performing a deprotection under the action of a cleavage solution to obtain semaglutide; the method is simple in operation, reduces side reactions, greatly reduces the racemization risk of His in an amino acid, and is convenient for large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of chemical synthesis, specifically relating to a method for synthesizing acylated GLP-1 compounds. Background Technology

[0002] Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder caused by various factors that impair the function of pancreatic β-cells, leading to insufficient insulin secretion or increased insulin resistance in target tissues. Clinically, it mainly manifests as polydipsia, polyphagia, polyuria, and weight loss, and carries the risk of complications such as sudden diabetic ketoacidosis and hyperosmolar coma. T2DM is a chronic metabolic disease that typically develops after age 35-40, accounting for over 90% of diabetes cases. Currently, clinical treatments for diabetes primarily focus on various types of insulin and four main classes of oral hypoglycemic agents. To provide patients with more treatment options, some new diabetes medications emerged in the early 21st century, such as glucagon-like peptide-1 (GLP-1) analogs, which are structurally modified versions of endogenous GLP-1. Endogenous GLP-1 is an incretin hormone secreted by L cells in the intestine. Containing over 30 amino acids, it binds to GLP-1 receptors widely distributed on the cell membranes of various organs and tissues, including the heart, liver, kidneys, pancreas, and gastrointestinal tract. This triggers changes in related intracellular signal transduction pathways, thereby regulating bodily functions. Therefore, GLP-1 exhibits multifunctional and multi-target effects, particularly significantly regulating pancreatic islet cells and promoting insulin secretion on demand based on blood glucose levels. However, its only drawback is that GLP-1 is readily degraded by dipeptidyl peptidase IV in the human body, with a half-life of approximately 2 minutes, which limits its clinical application. Structurally modified GLP-1 formulations retain the pharmacological effects of GLP-1 while significantly extending its duration of action.

[0003] Semaglutide is a novel long-acting GLP-1 receptor agonist developed by Novo Nordisk. It requires only once-weekly subcutaneous injection and is the second GLP-1 hypoglycemic agent in Novo Nordisk's diabetes pipeline to offer both blood sugar lowering and weight loss benefits (the first being liraglutide). Semaglutide's chemical name is N-ε. 26 -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetamido)ethoxy]ethoxy)acetyl][Aib8,Arg34]GLP-1-(7-37)peptide, molecular formula is C 187 H 291 N 45 O 59Its corresponding molecular weight is 4113.64, and its specific structure is shown in the following formula. The main peptide chain structure of smegglutinin is an intermediate polypeptide Arg34GLP-1(9-37) containing 29 amino acids, which can be obtained by solid-phase synthesis or gene recombination. At position 26, lysine (Lys) of smegglutinin is linked to a side chain composed of two aminoethoxyethoxyacetic acid (AEEA), glutamic acid, and octadecanoic acid fatty chains. At the same time, position 8 is replaced by the non-natural amino acid aminoisobutyric acid. Compared with liraglutinin, smegglutinin has a longer fatty chain and increased hydrophobicity. However, smegglutinin is also modified with short-chain AEEA, which greatly enhances its hydrophilicity. After AEEA modification, it can not only bind tightly to albumin and mask the hydrolytic site of DPP-4 enzyme, but also reduce renal excretion, prolong the biological half-life, and achieve the effect of long circulation.

[0004]

[0005] Currently reported patents related to the preparation of smegglutide include CN111378028A, CN105154498A, and CN113801233A. Among them, patent CN111378028A discloses that the two carboxyl groups on the side chain are exposed and not protected by protecting groups. When it is coupled with the Arg34GLP-1(9-37) main peptide chain in the subsequent reaction, the purity of the intermediate obtained is not high, only 67.5%, which seriously reduces the yield. Moreover, in this step of the reaction, pH has a significant impact on the side chain modification, which easily generates a variety of difficult-to-remove byproducts, such as double-modified impurity byproducts modified at both the 26 and terminal positions of the main peptide chain and byproducts modified at the terminal position of the main peptide chain. Compound 1 used in this patent is not easy to obtain (the synthesis steps are relatively more numerous and the cost is higher) and has low purity (in the previous step of preparing compound 1 from compound 6, N-succinimide is easily removed during the removal of tert-butyl group). Example 10 of patent CN105154498A discloses that the Arg34GLP-1(9-37) main peptide chain is first coupled to a side chain with a carboxyl group protected by tert-butyl (tBu), and then coupled to a Boc-His(Boc)-Aib-OH dipeptide in the presence of HATU and TEA. The reaction time is as long as 14 days. After post-processing HPLC purification, the freeze-drying time is as long as 16 hours, which is not conducive to industrial production. Moreover, the UV absorption of Boc is not as strong as that of Trt, which is not conducive to reaction monitoring. Furthermore, the stability of Boc is not as good as that of Trt, resulting in the generation of racemic impurities. Patent CN113801233A discloses a process in which the Boc-modified semaglutide precursor is first coupled with an Fmoc complex (such as Fmoc-His-Aib or Fmoc-His-Aib-Glu), then the Boc is removed from the tert-butyl-protected side chain, followed by the removal of the Fmoc, and finally the removal of the tert-butyl to obtain semaglutide. The entire process involves many steps and requires frequent deprotection (3 deprotection steps), which can lead to the racemization of some amino acids, especially the racemization of the terminal His and the introduction of other impurities due to incomplete Fmoc removal, making subsequent purification quite difficult.

[0006] Given the aforementioned technical problems with existing technologies, there is an urgent need to find a synthetic method for smegglutinin that is simple to operate, has low synthesis cost, higher efficiency, higher product purity, and is more suitable for industrial production. Summary of the Invention

[0007] The purpose of this invention is to overcome the above-mentioned shortcomings of the prior art. This invention provides a novel method for synthesizing smegglutinin, which is simple to operate, has low synthesis cost, higher efficiency, can realize industrial production, and produces products with higher purity and stable quality.

[0008] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0009] (1) Preparation of compound 2: Arg34GLP-1(9-37) was used as the starting material. Compound 1 was used to modify the side chain at the 26-position Lys of Arg34GLP-1(9-37). An acylation coupling reaction was carried out under certain reaction pH conditions. After post-treatment, the isoelectric point of the reaction solution was adjusted to precipitate a solid and obtain compound 2.

[0010] (2) Preparation of compound 4: The activated compound 3 and the Arg34GLP-1(9-37) terminal amino group in compound 2 were subjected to an acylation coupling reaction under the action of a condensing agent and a base to obtain compound 4.

[0011] (3) Preparation of smegglutinin: using compound 4 as raw material, smegglutinin was obtained by deprotection under the action of lysis buffer.

[0012] The specific preparation route I is shown below:

[0013]

[0014] In compounds 1, 2 and 4, the substituent R is a hydrocarbon group such as an alkane, alkene, alkyne or aromatic hydrocarbon.

[0015] In one embodiment of the present invention, R in compounds 1 and 4 is methyl, ethyl, tert-butyl, or benzyl.

[0016] In one embodiment of the present invention, R in compounds 1 and 4 is tert-butyl, and the specific synthetic route I' is as follows:

[0017]

[0018] In compounds 1, 2 and 4, the substituent R is tert-butyl.

[0019] In one embodiment of the present invention, the reaction pH in step (1) is 10-12, and the pH of the isoelectric point of the reaction solution is adjusted to 4-6.

[0020] In one embodiment of the present invention, the reagent used to control the pH value of the reaction in step (1) is a solution composed of alkali-water-organic solvent. The alkali is selected from organic alkali or inorganic alkali. The organic alkali is selected from any one of triethylamine, N,N-diisopropylethylamine, pyridine, piperidine, and aniline. The inorganic alkali is selected from any one of alkali metal hydroxides and alkali metal carbonates, such as sodium hydroxide, potassium hydroxide, sodium hydrogen phosphate, and potassium hydrogen phosphate. The organic solvent is selected from any one of acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, N,N-dimethylacetamide, and tetrahydrofuran.

[0021] In one embodiment of the present invention, the alkali-water-organic solvent is selected from any one of the following systems: triethylamine / water / acetonitrile system, N,N-diisopropylethylamine / water / acetonitrile system, N,N-diisopropylethylamine / water / N,N-dimethylformamide system, and N,N-diisopropylethylamine / water / N,N-dimethylacetamide system.

[0022] In one embodiment of the present invention, the mass ratio of water to organic solvent in the reagent used to control the pH value of the reaction in step (1) is 1-5:1-5.

[0023] In one embodiment of the present invention, the condensing reagent used in the coupling reaction in step (2) is any one or a combination of several of DCC (dicyclohexylcarbodiimide), DIC (N,N'-diisopropylcarbodiimide), HOSu (N-hydroxysuccinimide), HOBt (1-hydroxybenzotriazole), and p-nitrophenol.

[0024] In one embodiment of the present invention, the base used in the coupling reaction in step (2) is an organic base or an inorganic base. The organic base is any one of triethylamine, N,N-diisopropylethylamine, pyridine, piperidine, and aniline. The inorganic base is an alkali metal hydroxide or alkali metal carbonate, such as any one of sodium hydroxide, potassium hydroxide, sodium hydrogen phosphate, and potassium hydrogen phosphate.

[0025] In one embodiment of the present invention, the acid used to adjust the pH of the isoelectric point of the reaction solution in step (1) is an organic acid or an inorganic acid, wherein the organic acid is any one of trifluoroacetic acid, acetic acid, and formic acid; and the inorganic acid is any one of hydrochloric acid, phosphoric acid, and nitric acid.

[0026] In one embodiment of the present invention, the lysis solution in step (3) is any two or more combinations of trifluoroacetic acid, trifluoroethanol, benzyl sulfide, 1,2-ethanedithiol, phenol, triisopropylsilane, and water.

[0027] Compared with the prior art, the present invention has the following advantages: (1) The present invention controls the order of coupling reactions, that is, the main peptide chain is first coupled with the side chain with tert-butyl protection of the carboxyl group, then coupled with the Boc-His(Trt)-Aib-OH dipeptide, and finally removes the two protecting groups Boc and t-Bu in one step. The whole process only requires 3 steps to obtain smegglutinin with a purity ≥97%. (2) When the main peptide chain is coupled with the side chain with tert-butyl protection of the carboxyl group, compared with the process of directly reacting with the side chain without tert-butyl protection, the reaction time of the present invention is greatly shortened, the purity of the intermediate compound 2 obtained is higher, and the residual amount of by-products such as raw material Arg34GLP-1(9-37) and the content of double-modified impurities are far lower than the values ​​reported in the prior art. The higher the purity of the intermediate, the fewer impurities are introduced into the final product, especially the raw material Arg34GLP-1(9-37) and double-modified impurities that are more difficult to remove. (3) The dipeptide Boc-His(Trt)-Aib-OH is used, which, compared to dipeptides protected by Boc on Fmoc or His, can better avoid amino acid racemization, has better stability, is conducive to reaction monitoring, and eliminates the need for pre-protection of other side chain groups; (3) The N,N-diisopropylethylamine and Arg34GLP-1(9-37) raw materials are dissolved in a mixture of acetonitrile and water, and the pH is adjusted by isoelectric point precipitation in the post-treatment, which is simple to operate and has a low cost. (4) The entire process only involves deprotection in the last step, without the need for frequent deprotection, so it basically does not lead to amino acid racemization or the introduction of other impurities. In summary, the present invention has simple steps, convenient operation, low synthesis cost, higher efficiency, stable product quality, greatly reduces the risk of His racemization in amino acids, and can realize industrial production. Attached Figure Description

[0028] Figure 1 This is the liquid chromatogram of compound 2 obtained in Example 1.

[0029] Figure 2 This is the mass spectrum of compound 2 obtained in Example 1.

[0030] Figure 3 This is the liquid chromatogram of compound 2 obtained in Example 2.

[0031] Figure 4 This is the liquid chromatogram of compound 2 obtained in Example 3.

[0032] Figure 5 This is the liquid chromatogram of compound 2 obtained in Example 4.

[0033] Figure 6 This is the liquid chromatogram of compound 4 obtained in Example 5.

[0034] Figure 7 This is the liquid chromatogram of compound 4 obtained in Example 6.

[0035] Figure 8 This is the liquid chromatogram of compound 4 obtained in Example 7.

[0036] Figure 9 This is the liquid chromatogram of compound 4 obtained in Example 8.

[0037] Figure 10 This is the liquid chromatogram of smegglutinin obtained in Example 9.

[0038] Figure 11 This is the mass spectrum of smegglutinin obtained in Example 9.

[0039] Figure 12 This is the liquid chromatogram of smegglutinin obtained in Example 10.

[0040] Figure 13 This is the liquid chromatogram of smegglutinin obtained in Example 11.

[0041] Figure 14 This is the liquid chromatogram of smegglutinin obtained in Example 12.

[0042] Figure 15 Liquid chromatogram of smegglutinin obtained in Comparative Example 1

[0043] Figure 16 Liquid chromatogram of smegglutinin obtained in Comparative Example 2

[0044] Figure 17 Liquid chromatogram of smegglutinin obtained in Comparative Example 3

[0045] Figure 18 Liquid chromatogram of smegglutinin obtained in Comparative Example 4 Detailed Implementation

[0046] To facilitate understanding of the present invention by those skilled in the art, the technical solution of the present invention will be further described below in conjunction with specific embodiments, but the following content should not limit the scope of protection claimed in the claims of the present invention in any way.

[0047] Unless otherwise specified, all raw materials and reagents used in the following examples are commercially available.

[0048] The Arg34GLP-1(9-37) peptide chain was manufactured in-house by Nanjing Hanxin Pharmaceutical Technology Co., Ltd., and its preparation process was based on the publicly disclosed invention patent CN202111664146.2 filed by our company, the entire contents of which can be directly incorporated into this invention. Compound 1 (side chain), Boc-His(Trt)-Aib-OH (dipeptide), and other conventional reagents can all be obtained through commercial means.

[0049] The specific synthesis route is shown in Route I' published above.

[0050] Example 1

[0051] Arg34GLP-1(9-37) is modified with 26-bit Lys:

[0052] N,N-diisopropylethylamine was added to 300 mL of a mixture of acetonitrile and water (acetonitrile:water = 1:2), cooled to 0–5 °C, and the pH was adjusted to 11.5. 1 g of Arg34 GLP-1 (9-37) was added to form a mixture. 0.33 g of compound 1 was dissolved in 100 mL of acetonitrile and added dropwise to the above mixture. The reaction was carried out at 0–5 °C for 3 hours. The pH of the resulting reaction solution was adjusted to 5.4 with acetic acid, and then 400 mL of purified water was added. Centrifugation yielded solid compound 2. Figure 1 The liquid chromatography spectrum showed that the purity of compound 2 was 92.97%, the residual amount of the raw material Arg34GLP-1(9-37) was 3.74%, and no double-modified impurities were detected. Figure 2 The mass spectrum (MS) showed that the molecular weight of compound 2 was: MS: m / z = 4003.17 (M+H). + )Da.

[0053] Example 2

[0054] Arg34GLP-1(9-37) is modified with 26-bit Lys:

[0055] Triethylamine was added to 300 mL of a mixture of acetonitrile and water (acetonitrile:water = 1:1), the temperature was lowered to 0–5 °C, the pH was adjusted to 11.5, and 1 g of Arg34GLP-1 (9-37) was added to form a mixture. 0.33 g of compound 1 was dissolved in 100 mL of acetonitrile and added dropwise to the above mixture. The reaction was carried out at 0–5 °C for 3 hours. The pH of the resulting reaction solution was adjusted to 4.0 with hydrochloric acid, and then twice the volume of purified water was added. Centrifugation yielded solid compound 2. Figure 3 The liquid chromatography spectrum showed that the purity of compound 2 was 92.44%, the residual amount of raw material Arg34GLP-1(9-37) was 4.59%, and no double-modified impurities were detected.

[0056] Example 3

[0057] Arg34GLP-1(9-37) is modified with 26-bit Lys:

[0058] Triethylamine was added to 300 mL of a mixture of acetonitrile and water (acetonitrile:water = 1:3), the temperature was lowered to 0–5 °C, the pH was adjusted to 11.5, and 1 g of Arg34GLP-1 (9-37) was added to form a mixture. 0.33 g of compound 1 was dissolved in 100 mL of acetonitrile and added dropwise to the above mixture. The reaction was carried out at 0–5 °C for 3 hours. The pH of the resulting reaction solution was adjusted to 5.0 with trifluoroacetic acid, and then twice the volume of purified water was added. Centrifugation yielded solid compound 2. Figure 4 The liquid chromatography spectrum showed that the purity of compound 2 was 92.00%, and the residual amount of the raw material Arg34GLP-1(9-37) was 0.89%.

[0059] Example 4

[0060] Arg34GLP-1(9-37) is modified with 26-bit Lys:

[0061] Triethylamine was added to 300 mL of a mixture of acetonitrile and water (acetonitrile:water = 1:5), the temperature was lowered to 0–5 °C, the pH was adjusted to 11.5, and 1 g of Arg34GLP-1 (9-37) was added to form a mixture. 0.33 g of compound 1 was dissolved in 100 mL of acetonitrile and added dropwise to the above mixture. The reaction was carried out at 0–5 °C for 3 hours. The pH of the resulting reaction solution was adjusted to 6.0 with phosphoric acid, then twice the volume of purified water was added, and centrifugation was performed to obtain solid compound 2. Figure 5 The liquid chromatography spectrum showed that the purity of compound 2 was 91.42% and the residual amount of the raw material Arg34GLP-1(9-37) was 2.08%.

[0062] Example 5

[0063] Arg34GLP-1(9-37) was used for terminal amino group modification:

[0064] Dissolve 1g of Boc-His(Trt)-Aib-OH in 10mL of dichloromethane, add 0.37g of DCC, cool to 0℃, stir for 30min, add 0.22g of HOSu, stir and react at this temperature for 12 hours, filter, collect the filtrate, and evaporate the filtrate to dryness to obtain crude Boc-His(Trt)-Aib-OSu.

[0065] 0.33 g of the crude Boc-His(Trt)-Aib-OSu product and 1 g of compound 2 obtained in Example 1 were dissolved in 20 mL of N,N-dimethylformamide, and 2 mL of N,N-diisopropylethylamine were added. The mixture was reacted at room temperature for 8 h to obtain compound 4 with a purity of 76.57%. The specific liquid chromatography spectrum is shown below. Figure 6 As shown.

[0066] Example 6

[0067] Arg34GLP-1(9-37) was used for terminal amino group modification:

[0068] Dissolve 1 g of Boc-His(Trt)-Aib-OH in 10 mL of dichloromethane and add 0.37 g of DIC. Cool to 0 °C, stir for 30 min, add 0.22 g of HOSu, stir and react at this temperature for 12 hours, filter, collect the filtrate, and evaporate the filtrate to dryness to obtain crude Boc-His(Trt)-Aib-OSu.

[0069] 0.33 g of the crude Boc-His(Trt)-Aib-OSu product and 1 g of compound 2 obtained in Example 2 were dissolved in 20 mL of N,N-dimethylformamide, and 2 mL of N,N-diisopropylethylamine were added. The mixture was reacted at room temperature for 8 h to obtain compound 4 with a purity of 76.13%. The specific liquid chromatography spectrum is shown below. Figure 7 As shown.

[0070] Example 7

[0071] Arg34GLP-1(9-37) was used for terminal amino group modification:

[0072] Dissolve 1g of Boc-His(Trt)-Aib-OH in 10ml of dichloromethane and add 0.37g of HOSu. Cool to 0℃, stir for 30min, add 0.22g of HOSu, stir and react at this temperature for 12 hours, filter, collect the filtrate, and evaporate the filtrate to dryness to obtain crude Boc-His(Trt)-Aib-OSu.

[0073] 0.33 g of the crude Boc-His(Trt)-Aib-OSu product and 1 g of compound 2 obtained in Example 3 were dissolved in 20 mL of N,N-dimethylformamide, and 2 mL of N,N-diisopropylethylamine was added. The mixture was reacted at room temperature for 8 h to obtain compound 4 with a purity of 74.08%. The specific liquid chromatography spectrum is shown below. Figure 8 As shown.

[0074] Example 8

[0075] Arg34GLP-1(9-37) was used for terminal amino group modification:

[0076] Dissolve 1 g of Boc-His(Trt)-Aib-OH in 10 mL of dichloromethane, and add 0.34 g of p-nitrophenol. Cool to 0 °C, stir for 30 min, and add 0.5 g of DIC and 0.22 g of HOSu in portions. Stir and react at this temperature for 12 hours. Filter, collect the filtrate, and evaporate the filtrate to dryness to obtain crude Boc-His(Trt)-Aib-OSu.

[0077] 0.33 g of the crude Boc-His(Trt)-Aib-OSu product and 1 g of compound 2 obtained in Example 3 were dissolved in 20 mL of N,N-dimethylacetamide, and 2 mL of N,N-diisopropylethylamine were added. The mixture was reacted at room temperature for 8 h to obtain compound 4 with a purity of 77.23%. The specific liquid chromatography spectrum is shown below. Figure 9 As shown.

[0078] Example 9

[0079] Synthesis of smegglutinin:

[0080] 100 mg of compound 4, obtained in Example 5 with a purity of approximately 76.57%, was added to 1 mL of freshly prepared, chilled lysis buffer (trifluoroacetic acid: anisole: triisopropylsilane = 95:2:3). The mixture was reacted at room temperature for 3 h. Then, 10 mL of tert-methyl ether was added, precipitating a white solid. After centrifugation, the product was washed three times with tert-methyl ether, dried, and then purified by reversed-phase HPLC to obtain 90 mg of smegglutinin with a purity of 98.54%. The HPLC chromatogram is shown below. Figure 10 As shown, the mass spectrum is as follows Figure 11 As shown, the molecular weight of smegglutinin is: MS: m / z = 4113.16 (M+H) + ).

[0081] Example 10

[0082] Synthesis of smegglutinin:

[0083] 100 mg of compound 4, obtained in Example 6 with a purity of approximately 76.13%, was added to 1 mL of freshly prepared, chilled lysis buffer (trifluoroacetic acid: 1,2-ethylenedithiol: triisopropylsilane = 95:2:3). The mixture was reacted at room temperature for 3 h. Then, 10 mL of methyl ether was added, precipitating a white solid. After centrifugation, the product was washed three times with methyl ether, followed by two washes with 10 mL of ethyl acetate. After drying, 85 mg of smegglutinin with a purity of 98.43% was obtained by reversed-phase HPLC purification. The HPLC chromatogram is shown below. Figure 12 As shown.

[0084] Example 11

[0085] Synthesis of smegglutinin:

[0086] 100 mg of compound 4, obtained in Example 7 with a purity of approximately 74.08%, was added to 1 mL of freshly prepared, chilled lysis buffer (trifluoroacetic acid: water: triisopropylsilane = 95:2:3). The mixture was reacted at room temperature for 3 h. Then, 10 mL of ice-cold methyl ether was added, precipitating a white solid. After centrifugation, the product was washed three times with methyl ether, dried, and then purified by reversed-phase HPLC to obtain 90 mg of smegglutinin with a purity of 97.27%. The specific HPLC chromatogram is shown below. Figure 13 As shown.

[0087] Example 12

[0088] Synthesis of smegglutinin:

[0089] 100 mg of compound 4, obtained in Example 8 with a purity of approximately 77.23%, was added to 1 mL of freshly prepared, chilled lysis buffer (trifluoroacetic acid: phenol: triisopropylsilane = 95:2:3). The mixture was reacted at room temperature for 3 h. Then, 10 mL of methyl ether was added, precipitating a white solid. After centrifugation, the product was washed three times with methyl ether, followed by two washes with 10 mL of ethyl acetate. After drying, 84 mg of smegglutinin with a purity of 97.52% was obtained by reversed-phase HPLC purification. The specific HPLC chromatogram is shown below. Figure 14 As shown.

[0090] Comparative Example 1

[0091] Referring to the method disclosed in patent CN111378028A, the reaction formula is shown below. The main difference is that the side chain of compound 1 does not have a protecting group, and the other conditions are the same as in Example 1.

[0092]

[0093] Arg34GLP-1(9-37) is modified with 26-bit Lys:

[0094] N,N-diisopropylethylamine was added to a 300 mL mixture of acetonitrile and water, cooled to 0–5 °C, and the pH was adjusted to 11.5. 1 g of Arg34GLP-1 (9-37) was added to form a mixture. 0.29 g of compound 1' was dissolved in 100 mL of acetonitrile and added dropwise to the above mixture. The reaction was carried out at 0–5 °C for 1 hour. The pH of the resulting reaction solution was adjusted to 5.4 with acetic acid, and then 400 mL of purified water was added. Centrifugation yielded a solid compound 2' with a purity of only 75.83%. The specific liquid chromatogram is shown below. Figure 15 As shown.

[0095] A comparison between Example 1 and Comparative Example 1 shows that by replacing the raw materials, the quality and purity of compound 2' obtained by the process in Comparative Example 1 are significantly reduced (purity decreased by 17%). The purity of the intermediate compound 2' is 75.83%, and the amount of by-products increases significantly. For example, the residual amount of raw material Arg34GLP-1(9-37) is 18.35% (an order of magnitude higher than the impurities in Example 1), and the content of dual-modified impurities is 1.24%, which is much higher than the values ​​in Example 1. Specifically, as shown in the figure... Figure 15 As shown, the lower the purity of the intermediate, the more impurities are introduced into the intermediate or product, especially the more difficult-to-remove raw material Arg34GLP-1(9-37) and double-modified impurities.

[0096] Comparative Example 2

[0097] Referring to the method of patent CN105154498A, Boc-His(Boc)-Aib-OH dipeptide was used instead of Boc-His(Trt)-Aib-OH dipeptide in this invention, and all other conditions were the same as in Example 6.

[0098] Specifically:

[0099] Arg34GLP-1(9-37) was used for terminal amino group modification:

[0100] Dissolve 1 g of Boc-His(Boc)-Aib-OH in 10 mL of dichloromethane and add 0.37 g of DIC. Cool to 0 °C, stir for 30 min, add 0.22 g of HOSu, stir and react at this temperature for 12 hours, filter, collect the filtrate, and evaporate the filtrate to dryness to obtain crude Boc-His(Trt)-Aib-OSu.

[0101] 0.33 g of the crude Boc-His(Boc)-Aib-OSu product and 1 g of compound 2 obtained in Example 2 were dissolved in 20 mL of N,N-dimethylformamide, and 2 mL of N,N-diisopropylethylamine were added. The mixture was reacted at room temperature for 8 h. The reaction was qualitatively detected using ninhydrin, and the result was positive. The reaction was continued for another 24 h, and the result became negative, yielding 1.08 g of compound 4 with a purity of 52.80%. The specific liquid chromatography chromatogram is shown below. Figure 16 As shown.

[0102] A comparison of Example 6 and Comparative Example 2 shows that using the Boc-His(Boc)-Aib-OSu dipeptide significantly increases the reaction time, reducing the conversion rate of the coupling reaction within the same timeframe. Furthermore, the UV absorption of Boc is not as strong as that of Trt, which is detrimental to reaction monitoring; and the stability of Boc is not as good as that of Trt, leading to the generation of racemic impurities.

[0103] Comparative Example 3

[0104] Referring to the method of patent CN113801233A, Fmoc-His-Aib-OH dipeptide is used instead of Boc-His(Trt)-Aib-OH dipeptide in this invention, and GLP-(9-37) requires that the amino group of Lys be protected with Boc beforehand.

[0105] The specific reaction formula is as follows:

[0106]

[0107] Arg34GLP-1(9-37) was used for terminal amino group modification:

[0108] Dissolve 1 g of Fmoc-His-Aib-OH in 10 mL of dichloromethane and add 0.37 g of DIC. Cool to 0 °C, stir for 30 min, add 0.22 g of HOSu, stir and react at this temperature for 12 hours, filter, collect the filtrate, and evaporate the filtrate to dryness to obtain crude Fmoc-His-Aib-OSu.

[0109] 0.33 g of the crude Fmoc-His-Aib-OSu and 1 g of Boc-protected Arg34GLP-1(9-37) were dissolved in 20 mL of N,N-dimethylformamide, and 2 mL of N,N-diisopropylethylamine was added. The mixture was reacted at room temperature for 8 h. 20 mL of a mixed solution of tertiary methyl ether and petroleum ether was added, and the precipitate was centrifuged to obtain Fmoc-GLP-1(Lys 20 The crude product (Boc) was added to a trifluoroacetic acid solution and stirred at low temperature for 2 hours. Then, 400 mL of methyl ether was added, and centrifugation yielded 0.98 g of solid. The crude product was dissolved in 20 mL of N,N-dimethylformamide, and 2 mL of N,N-diisopropylethylamine was added. 0.33 g of the side chain of compound 1 was dissolved in 2 mL of N,N-dimethylformamide and slowly added dropwise to the reaction solution. The reaction was carried out at room temperature for 2–3 hours. Then, a mixed solution of 400 mL of methyl ether and petroleum ether was added, and centrifugation yielded a solid. The solid was then added to 4 mL of 20% piperidine DMF solution and reacted at room temperature for 1 hour. Then, a mixed solution of 40 mL of methyl ether and petroleum ether was added, and centrifugation yielded a solid. Finally, 10 mL of cutting fluid (TFA:TIS:DCM = 95:3:2) was added, and the reaction was carried out at room temperature for 2–4 hours. Then, a mixed solution of 100 mL of methyl ether and petroleum ether was added, and centrifugation yielded 0.8 g of solid with a purity of 57.15%. The specific liquid chromatography spectrum is shown below. Figure 17 As shown.

[0110] A comparison between Example 6 and Comparative Example 3 shows that the steps in Comparative Example 3 are cumbersome. The Boc-modified semaglutide precursor is first coupled with the Fmoc complex (Fmoc-His-Aib), then the Boc is removed, coupled with the tert-butyl-protected side chain, then the Fmoc is removed, and finally the tert-butyl is removed to obtain semaglutide. The entire process requires five steps and frequent deprotection (three times), which can lead to racemization of certain amino acids and the formation of deletion peptides, especially the racemization of the terminal His and the introduction of other impurities from incomplete Fmoc removal, making subsequent purification more difficult. In addition, the semaglutide precursor requires protection, further increasing the complexity of the process.

[0111] Comparative Example 4

[0112] Arg34GLP-1(9-37) is modified with 26-bit Lys:

[0113] N,N-diisopropylethylamine was added to a 300 mL aqueous mixture, cooled to 0–5 °C, and the pH was adjusted to 11.5. 1 g of Arg34GLP-1 (9-37) was added to form a mixture. 0.33 g of compound 1 was dissolved in 100 mL of acetonitrile and added dropwise to the mixture. The reaction was carried out at 0–5 °C for 3 hours. Acetonitrile was removed by rotary evaporation. The resulting concentrate was diluted with water to a certain concentration, and the aqueous phase was freeze-dried. The purity of compound 2 was only 78.52%. The specific liquid chromatogram is shown below. Figure 18 As shown.

[0114] A comparison between Example 1 and Comparative Example 4 shows that Example 1 uses a mixed system of acetonitrile and water to dissolve N,N-diisopropylethylamine and Arg34GLP-1(9-37) raw materials. At the same time, the post-treatment uses acid to adjust the pH to 4-6, i.e., isoelectric point precipitation. The purity of the obtained compound 2 is as high as 90% or more, which is at least 10% higher than that of Comparative Example 4. At the same time, it eliminates the complicated procedures of rotary evaporation to remove organic solvents and subsequent freeze drying, and can greatly reduce costs.

Claims

1. A method for synthesizing smegglutinin, characterized in that, Includes the following steps: (1) Preparation of compound 2: Arg34GLP-1(9-37) was used as the starting material. Compound 1 was used to modify the side chain at the 26-Lys position of Arg34GLP-1(9-37). An acylation coupling reaction was carried out under certain reaction pH conditions. After post-treatment, the pH of the reaction solution was adjusted to 4-6, and a solid was precipitated to obtain compound 2. The reaction pH was 10-12. The reagent used to control the pH value of the reaction is a solution composed of alkali-water-organic solvent, wherein the alkali-water-organic solvent is selected from the triethylamine / water / acetonitrile system or the N,N-diisopropylethylamine / water / acetonitrile system; the mass ratio of water to organic solvent in the reagent used to control the pH value of the reaction is 1-5:1-5; (2) Preparation of compound 4: After activation, compound 3 was acylated and coupled with the Arg34GLP-1(9-37) terminal amino group in compound 2 under the action of condensing reagent and base to obtain compound 4. (3) Preparation of smegglutide: Using compound 4 as raw material, smegglutide is obtained by deprotection under the action of lysis buffer; the lysis buffer is selected from a combination of trifluoroacetic acid, anisole sulfide and triisopropylsilane or a combination of trifluoroacetic acid, 1,2-ethylenedithiol and triisopropylsilane. The specific preparation route is shown below: In compounds 1, 2 and 4, the substituent R is tert-butyl.

2. The synthesis method according to claim 1, characterized in that: The condensation reagent used in the coupling reaction in step (2) is any one or a combination of several of DCC, DIC, HOSu, HOBt or p-nitrophenol.

3. The synthesis method according to claim 1, characterized in that: The base used in the coupling reaction in step (2) is an organic base or an inorganic base. The organic base is any one of triethylamine, N,N-diisopropylethylamine, pyridine, piperidine, and aniline. The inorganic base is any one of alkali metal hydroxide and alkali metal carbonate.

4. The synthesis method according to claim 3, characterized in that: The inorganic base is any one of sodium hydroxide, potassium hydroxide, sodium hydrogen phosphate, and potassium hydrogen phosphate.

5. The synthesis method according to claim 1, characterized in that: The acid used to adjust the pH of the isoelectric point of the reaction solution in step (1) is an organic acid or an inorganic acid. The organic acid is any one of trifluoroacetic acid, acetic acid, and formic acid; the inorganic acid is any one of hydrochloric acid, phosphoric acid, and nitric acid.

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